Study population
To participate in the Houston Vanguard study, subjects had to be at least 18 years old, have documented HIV-1 infection and a CD4 cell count ≥ 350 cells/mm3 at screening. The Karnofsky performance status had to be ≥ 80 and the patients had to be receiving antiretroviral therapy for at least 7 days prior to starting scIL-2 therapy. Women of childbearing potential were required to have a negative pregnancy test before initiation of scIL-2 therapy. At baseline, all but one of the individuals had plasma HIV RNA levels below 500 copies/mL. That single individual had a viral load of 2,007 copies/mL. The main exclusion criteria for this study included any concurrent or prior history of AIDS-defining illnesses, malignancy requiring systemic therapy within the prior 5 years, concomitant use of systemic corticosteroids, chemotherapy or experimental cytotoxic drugs, or current or prior history of autoimmune and/or inflammatory diseases and breastfeeding. This study was approved by the institutional review board (IRB) at The University of Texas Health Science Center at Houston and the National Institute of Allergy and Infectious Disease (NIAID) IRB. All subjects provided written informed consent.
Trial design
The Houston Vanguard study was an open-label, randomized trial designed with a target sample size of 72 subjects with CD4 cell count change from baseline as the primary endpoint. Subjects were sequentially randomized to three groups of 24 (12 in each treatment group, scIL-2 or antiretroviral alone). The first group of 24 subjects received 1.5 Million International units (MIU) per dose of scIL-2 plus antiretroviral therapy or antiretroviral therapy alone (control); the second group of 24 subjects was randomized to 4.5 MIU scIL-2 or control; and the third group was randomized to 7.5 MIU of scIL-2 or control. For the purpose of this immunology sub-study of the Houston Vanguard, consecutive patients who volunteered to participate in the sub-study were enrolled: five patients from the 1.5 MIU per dose cohort (low dose), five patients from the 7.5 MIU per dose cohort (high dose) and four from the no IL-2 cohort (control group).
scIL-2 treatment
Subcutaneous IL-2 (aldesleukin [Proleukin]; Chiron, Emeryville, CA) was administered for 5 days every 8 weeks for a minimum of three cycles in addition to continued antiretroviral therapy. Dosing with scIL-2 was begun at 1.5 MIU. This dose was escalated to 4.5 MIU and then to 7.5 MIU when at least 9 of the 12 subjects had completed all doses of the first cycle with scIL-2 at a lower dose level in the absence of dose-limiting toxicities. Dose reductions of scIL-2 were allowed for dose-limiting toxicities defined as grade IV toxicities (as defined in the National Institutes of Health, Division of AIDS Toxicity Table for Grading Adverse Events), investigator's assessment or subject tolerance.
After completion of three cycles, subjects were enrolled in an extension phase (months 6 to 12) and, thereafter, rolled-over into the ESPRIT study (Evaluation of subcutaneous Proleukin® in a Randomized International Trial). Additional cycles of scIL-2 were encouraged to maintain CD4 cell counts more than twice baseline or greater than 1,000 cells/mm3. For subjects randomized to 1.5 and 4.5 MIU, dose escalation was allowed by increments of 1.5 MIU per dose up to a maximum of 7.5 MIU. Subjects receiving less than 4.5 MIU were permitted to dose escalate by increments of 1.5 to 3.0 MIU upon the investigator's discretion. Dosage could be reduced by 1.5 MIU decrements (to a minimum dose of 1.5 MIU) if the assigned dose was not tolerated. During the extension phase and roll-over ESPRIT no control subjects were treated with scIL-2 at any dose. The cumulative amount of IL-2 given to the 10 IL-2 treated patients in this study ranged between 165–570 MIU, with a mean of 302 ± 132.
Follow-up studies
Of the 14 originally enrolled in the study, 11 were still followed by Year 6. One patient with low CD8+ T cell apoptosis at baseline dropped out before Year 2, and two of the non-IL-2 patients withdrew consent, one before Year 1 and one before Year 3. Both of these patients had high levels of CD8+ T cell apoptosis at baseline.
Measurements of viral load/Lymphocyte aubsets
Plasma HIV RNA levels were quantified by branched DNA signal amplification assay (Quantiplex; Chiron Emeryville, CA, Version 2.0, lower limit of detection 500 copies/mL; and 3.0, lower limit of sensitivity 50 copies/mL). CD4 and CD8 counts were done by commercial flow cytometry laboratories using CAP defined criteria.
Measurements of activation and death on lymphocyte subpopulations
Peripheral blood was separated using Ficoll gradients. The cells were washed in PBS, counted and then stained for cell surface markers and Annexin V to determine levels of apoptotic cells. CD4, CD8, CD25, CD38, and DR antibodies were purchased from BD Pharmingen (San Jose, CA) and Annexin V was obtained from Biosource International (Camarillo, CA). The cells were analyzed using a Beckman Coulter XL2 flow cytometer. Twenty-thousand events were collected, gated on "viable" light scatter, and fluorescence distributions collected and analyzed and expressed as percentages or mean fluorescence intensity [18].
Statistical analysis
Group means and standard deviations of study variables: CD4, CD8, and CD4:CD8 ratios were obtained for both low and high annexin groups. Tests for normality were performed. Normally distributed data were analyzed using the t-test and one way repeated measures analysis of variance. A difference of p [22]. Variables entered, separately at first, in the models as covariates of annexin included age at randomization, age at diagnosis and race. CD4 nadir was not included because it is a component of the outcome variables (CD4 T cell count and CD4: CD8 ratio). Age was included as a continuous variable. Race was dichotomized to black and non-black.
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